Distribution of Karyotypes of the Cryptocercus Punctulatus Species Complex (Blattodea: Cryptocercidae) in Great Smoky Mountains National Park

Journal of Insect Science (2017) 17(3): 69; 1–11 doi: 10.1093/jisesa/iex045 Research article

Distribution of Karyotypes of the Cryptocercus punctulatus Species Complex (Blattodea: Cryptocercidae) in Great Smoky Mountains National Park

Christine A. Nalepa,1,2 Keisuke Shimada,3,4 Kiyoto Maekawa,3 and Peter Luykx5

1Department of Entomology, North Carolina State University, Campus Box 7613, Raleigh, NC 27695-7613, USA ([email protected]), 2Corresponding author, e-mail: [email protected], 3Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama, Toyama 930-8555, Japan ([email protected]; [email protected]), 4Current address: Ishikawa Museum of Natural History, Ri-441 Choshi-machi, Kanazawa, Ishikawa 920-1147, Japan and 5Department of Biology, University of Miami, Coral Gables, FL 33124, USA ([email protected])

Subject Editor: Evan Preisser

Received 3 February 2017; Editorial decision 11 April 2017

Abstract During the period between 1999 and 2006, wood-feeding cockroaches in the Cryptocercus punctulatus Scudder species complex were collected throughout Great Smoky Mountains National Park, USA. The chromosome numbers of insects from 59 sites were determined, and phylogenetic analyses were performed based on mitochondrial COII and nuclear ITS2 DNA. The distribution of the three male karyotypes found in the park (2n ¼ 37, 39, and 45) is mapped and discussed in relation to recent disturbances and glacial history. Clades of the three karyotype groups meet near the ridgeline separating North Carolina from Tennessee in the center of the park, suggesting that these may have originated from separate lower elevation refugia after the last glacial maximum. The timing of divergence and a signiﬁcant correlation between elevation difference and genetic distance in two of the clades supports this hypothesis. The ecological role of the cockroaches in the park is discussed.

Key words: All Taxa Biodiversity Inventory, refugia, ecological service, coarse woody debris

Great Smoky Mountains National Park (GSMNP) is home to one of eastern half of the park, and also at the eastern border near the oldest and most ecologically diverse mountain chains in the world, Waterville Lake. The Tennessee half of the park was largely un- and is an internationally recognized hotspot of temperate forest biodi- sampled. versity (Nichols and Langdon 2007). In an effort to catalogue this di- These karyotype groups have been described as separate species versity, a comprehensive inventory of all life forms in the GSMNP, the (Burnside et al. 1999). The validity of the proposed species-level sta- All Taxa Biodiversity Inventory (ATBI), was initiated and charged tus was questioned, however, because chromosome numbers were with the goal of discovering the identity and distribution of as many known for only part of the sample, the evolutionary relationships species as possible that occur in the park (Sharkey 2001, White and among members of the species complex were unclear, and although Langdon 2006). As part of this effort, the wingless wood-feeding cock- morphological variation was apparently present, it had not been roach Cryptocercus punctulatus Scudder was recovered from seven lo- demonstrated that this variation consistently distinguished the pro- cations in the park, representing seven watersheds (Discover Life in posed species (Nalepa et al. 2002, Everaerts et al. 2008, Maekawa America website, accessed 14 November 2016: https://www.dlia. and Nalepa 2011). Lineages that share a male chromosome number org/atbidata/MapTaxon.php?taxon¼Species&tname¼Cryptocercus_ may represent different species, subspecies, or races (Everaerts et al. punctulatus). Cryptocercus punctulatus in the eastern United States, 2008, Che et al. 2016). Consequently, Cryptocercus found along the however, is a cryptic-species complex currently divided into four Appalachian Chain in the eastern United States is best thought of as known karyotype groups, three of which have been reported from 12 a species complex that requires additional study before its taxonomy sites previously sampled in the park (Nalepa et al. 2002, Everaerts can be delineated. et al. 2008, Maekawa and Nalepa 2011). The karyotypes of males Despite the genetic differences among karyotype groups of C. consist of 18–22 pairs of autosomes and a single X chromosome. The punctulatus, there are no known differences in their ecology within 2n ¼ 37 and 2n ¼ 39 male karyotypes were found in the North their examined range; nor is there evidence of clear divergence in life Carolina section of the park; the 2n ¼ 45 karyotype was identified history, biology or behavior. All members of the genus are fairly along the crest separating North Carolina and Tennessee in the large, subsocial insects that are wingless and log-dependent; i.e., all

VC The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. 1 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 2 Journal of Insect Science, 2017, Vol. 17, No. 3 stages nest in coarse woody debris (CWD) that serves as both food Molecular Analysis and shelter. The distribution of the cockroach is therefore intimately DNA Extraction, Amplification, Purification, and Sequencing. tied to the distribution of their log hosts. Any event that has an im- Total DNA was extracted from the leg tissue of individuals pre- pact on mature forests, including deforestation and glaciation, will served in 80–100% ethanol by using DNeasy Tissue Kit (Qiagen, have an impact on the distribution of C. punctulatus (Nalepa 2001; Tokyo, Japan). Each individual was considered to be representative Nalepa et al. 2001, 2002). Because 95% of GSMNP is forested, of the population in each location. The fragments of mitochondrial with downed logs at all stages of decay on the forest floor (Martin COII (448 bp) and nuclear ITS2 (400 bp) were amplified using 1992, Sharkey 2001), the cockroach is expected to be more or less PCR. Primer sequences for the amplifications of COII and ITS2 are continuously distributed within the park. shown in Park et al. (2004) and Everaerts et al. (2008), respectively. The goal of this study is to begin documenting the spatial organi- The temperature profile for amplifications of COII and ITS2 was zation of the three known karyotypes of C. punctulatus within 94 C for 3 min, followed by 35 cycles of 94 C for 1 min, 50 C for GSMNP, and to determine whether the 2n ¼ 43 karyotype, not pre- 1 min and 70 C for 2 min. Amplified PCR products were purified viously detected in the park, can be found there. This information by using the Mag Extractor Kit (Toyobo, Osaka, Japan) or ExoSAP- would contribute to the existing ATBI database, and aid in the IT (Affymetrix, Santa Clara, CA, USA), and they were used as tem- search for concordant patterns of distribution in other taxa with low plates for sequencing performed by the DNA sequencer (ABI 373 or vagility and similar ecological requirements. If diverse organisms 3130 Genetic Analyzer; Applied Biosystems, Carlsbad, CA, USA). had retreated to and shared the same refugial areas during glacial cy- Sequence Alignments. For alignments, MUSCLE in MEGA ver- cles of the Pleistocene, some degree of geographic patterning would sion 6.06 (Tamura et al. 2013) was used. To consider the possibility be expected (Howden 1985, Vermeij 1986, Cranston and Naumann of mitochondrial introgression, we analyzed mitochondrial (COII) 1991, Soltis et al. 2006). Evidence from a variety of sources indi- and nuclear (ITS2) data separately. For the mitochondrial dataset, cates that there were one or more refugia in the Southern Cryptocercus clevelandi Byers was included as an outgroup Appalachians during the glacial cycles of the Pleistocene (Tilley (GenBank accession no. AB078557). For the nuclear dataset, how- 1997, Church et al. 2003, Soltis et al. 2006, Sokolov et al. 2007, ever, as shown in Everaerts et al. (2008), there were many insertions Walker et al. 2009, Rissler and Smith 2010, Garrick 2011). and deletions in DNA sequences between C. clevelandi and cockroaches from the southern Appalachians. Thus, the ITS2 tree is an Materials and Methods unrooted tree. Phylogenetic Analysis. We obtained estimations of tree topolo- Insect Sampling gies under the Bayesian inference (BI), maximum likelihood (ML) Cryptocercus cockroaches were collected between 1999 and 2006, and maximum parsimony (MP) methods. For BI, the most appropri- in some cases in conjunction with other studies of the genus. Four ate model of sequence evolution was determined using MEGA ver- collection sites were previously reported in Nalepa et al. (2002) and sion 6.06 model selection option (Tamura et al. 2013). The T92 þ G eight additional sites were detailed in Everaerts et al. (2008) (see model was selected from both COII and ITS2 data. Parameters for Table 1). Some location-coordinates may have shifted slightly from the selected model of substitution were estimated from the data. In those originally in the literature; more precise location data have total, 100,000 trees were obtained (ngen ¼ 10,000,000, become available as technology has advanced. Here we combine samplefreq ¼ 100) using MrBayes version 3.2.6 (Ronquist and data on those 12 sites with data collected on cockroaches from an Huelsenbeck 2003), and the first 25% of these (25,000) were dis- additional 47 sampling locations within the park (total ¼ 59) to give carded as the burn-in. A 50%-majority-rule consensus tree of the a more comprehensive summary of the geographic distribution of remaining trees was produced. Two independent runs under the the different karyotypes within the park. The samples include three same model of sequence evolution were performed. For ML, 1,000 sites from the Foothills Parkway (sites #81–83) and two sites just bootstrap replicates were performed based on the same model of outside park boundaries (sites #39, 85). sequence evolution as BI in MEGA 6.06 (Tamura et al. 2013). At each site, cockroaches were sampled from rotting logs until at Initial trees for the heuristic search were obtained by applying the least three adult males were collected for chromosome preparations. NJ method to a matrix of pairwise distances estimated using the In some locations these males were all from a single log. At other Maximum Composite Likelihood approach. For MP analysis, all times more logs had to be sampled; however, the sampled logs were characters were included and weighted equally, and 1,000 bootstrap 2 always located within an area of 50 m . In a few cases (sites #126, replicates were performed using MEGA version 6.06 (Tamura et al. 127, 128, 141), the karyotype and molecular analyses were done 2013). Subtree-Pruning-Regrafting algorithm with search level 1, in using the same male specimen; in most, however, females and which the initial trees were obtained by the random addition of nymphs from the same collections were used for molecular analyses. sequences (10 replicates), was used. Collections were limited to locations reached by round-trip hiking Estimation of Divergence. As in previous studies of Korean in one day, so there is some bias associated with accessibility of the Cryptocercus spp. (Park et al. 2004), we did a preliminary estima- sampling sites. The more interior locations were under-sampled, tion of divergence times between each phylogenetic group (I–VI in particularly in the western half of the park. Figs. 1 and 2) based on the COII sequences using transversion (TV) Cockroaches were classified by chromosome number without distances (0.13–0.30%/million years (Myr)). Mean TV distances implications of taxonomic status, and were collected under permits were calculated as the Jukes–Cantor formula modified by GRSM-99-063, GRSM-2001-SCI-0024, and GRSM-2005-SCI-0084. Beckenbach et al. (1993).

Chromosome Counts For each sampled location, chromosome preparations were made Principal Component Analysis (PCA) from the testes of three adult males using the technique of Luykx To understand the correlation of the chromosome numbers and phy- (1983). Meiotic chromosomes were counted and are reported as the logenetic groups with the sampling location (latitude, longitude and male diploid count. elevation shown in Table 1), we did a PCA using the statistical Journal of Insect Science, 2017, Vol. 17, No. 3 3

Table 1. Collection localities, chromosome number of males, and Genbank accession numbers of Cryptocercus collected in Great Smoky Mountains National Park

Collection Site Location Male 2n County Latitude Longitude Elev. (m) Published? COII ITS2 no. no. Accession no. Accession no.

1 49 Wolf Ridge 37 Swain, NC 36.475 83.880 480 Nalepa et al. 2002 LC218170 LC218221 2 50 Thomas Divide 37 Swain, NC 35.466 83.419 666 Nalepa et al. 2002 LC218171 LC218222 3 81 Chilhowee 37 Blount, TN 35.559 84.011 317 LC218172 LC218223 4 82 Look Rock 37 Blount, TN 35.633 83.942 797 LC218173 LC218224 5 83 Mt. Nebo 37 Blount, TN 35.717 83.821 360 LC218174 LC218225 6 84 Cades Cove 37 Blount, TN 35.607 83.779 627 LC218175 LC218226 7 85 Townsend 37 Blount, TN 35.668 83.716 398 LC218176 LC218227 8 86 Elkmont 37 Sevier, TN 35.663 83.600 629 LC218177 LC218228 9 87 Chimneys 37 Sevier, TN 35.636 83.493 837 LC218178 LC218229 10 88 Greenbrier 37 Sevier, TN 35.733 83.415 438 LC218179 LC218230 11 91 Clingmans Dome 37 Swain, NC 35.558 83.493 1,895 LC218180 LC218231 12 109 Ramsay Cascades 37 Sevier, TN 35.710 83.319 856 LC218181 LC218232 13 111 Maddron Bald Trail 37 Cocke, TN 35.769 83.267 549 LC218182 LC218233 14 113 Collins Creek Trail 37 Swain, NC 35.569 83.339 742 LC218183 LC218234 15 122 Noland Divide Trail 37 Swain, NC 35.566 83.475 1,742 Everaerts et al. 2008 AB425865 AB425887 16 123 Keg Drive Branch 37 Swain, NC 35.577 83.449 1,480 Everaerts et al. 2008 AB425866 AB425888 17 124 Fork Ridge Trailhead 37 Swain, NC 35.590 83.470 1,794 LC218184 LC218235 18 129 Deep Low Gap 37 Swain, NC 35.512 83.348 1,103 Everaerts et al. 2008 AB425864 AB425886 19 130 Mingus Mill 37 Swain, NC 35.520 83.309 597 LC218185 LC218236 20 147 Old Sugarlands 37 Sevier, TN 35.674 83.493 686 LC218186 LC218237 21 153 Roaring Fork 37 Sevier, TN 35.681 83.462 768 LC218187 LC218238 22 51 Kephart Prong Trail 39 Swain, NC 35.590 83.371 897 Nalepa et al. 2002 LC218188 LC218239 23 93 Smokemont 39 Swain, NC 35.556 83.310 711 LC218189 LC218240 24 94 Palmer Creek 39 Haywood, NC 35.628 83.175 1,377 LC218190 LC218241 25 95 Straight Fork 39 Swain, NC 35.581 83.243 815 LC218191 LC218242 26 98 Cataloochee Divide 39 Haywood, NC 35.632 83.045 1,251 LC218192 LC218243 27 106 Sunkota Ridge 39 Swain, NC 35.546 83.369 1,442 LC218193 LC218244 28 107 Kanati Fork Trail 39 Swain, NC 35.572 83.385 1,505 LC218194 LC218245 29 108 Thomas Divide Trailhead 39 Swain, NC 35.585 89.399 1,417 LC218195 LC218246 30 112 Deep Creek Trailhead 39 Swain, NC 35.600 83.424 1,416 LC218196 LC218247 31 140 Kephart Shelter 39 Swain, NC 35.611 83.369 1,078 LC218197 LC218248 32 141 Cabin Flats 39 Swain, NC 35.609 83.333 891 Everaerts et al. 2008 AB425871 AB425893 33 39 Waterville Lake 45 Haywood, NC 35.699 83.041 867 Nalepa et al. 2002 LC218198 LC218249 34 89 Cosby 45 Cocke, TN 35.783 83.217 517 LC218199 LC218250 35 90 Big Creek 45 Haywood, NC 35.766 83.109 507 LC218200 LC218251 36 92 Newfound Gap 45 Sevier, TN 35.611 83.424 1,534 LC218201 LC218252 37 114 Sweat Heifer 45 Sevier, TN 35.621 83.404 1,777 Everaerts et al. 2008 AB425882 AB425904 38 115 Boulevard Trail 45 Sevier, TN 35.630 83.392 1,853 LC218202 LC218253 39 116 Anakeesta Knob 45 Sevier, TN 35.637 83.411 1,792 LC218203 LC218254 40 117 West Point View 45 Sevier, TN 35.652 83.444 1,932 LC218204 LC218255 41 118 Arch Rock 45 Sevier, TN 35.636 83.439 1,439 LC218205 LC218256 42 119 Grassy Patch 45 Sevier, TN 35.629 83.449 1,250 LC218206 LC218257 43 125 Road Prong 45 Sevier, TN 35.610 83.448 1,618 LC218207 LC218258 44 126 Mt. Collins 45 Sevier, TN 35.595 83.476 1,698 Everaerts et al. 2008 AB425881 AB425903 45 127 Indian Grave Flats 45 Sevier, TN 35.620 83.470 1,235 LC218208 LC218259 46 128 Chimney Tops 45 Sevier, TN 35.634 83.470 1,024 LC218209 LC218260 47 134 Camelback 45 Cocke, TN 35.726 83.207 1,428 Everaerts et al. 2008 AB425884 AB425906 48 135 Upper Low Gap 45 Cocke, TN 35.737 83.182 1,293 LC218210 LC218261 49 136 Cosby Creek 45 Cocke, TN 35.753 83.206 671 LC218211 LC218262 50 137 Mt. Sterling 45 Haywood, NC 35.700 83.098 1,134 LC218212 LC218263 51 138 Double Gap 45 Haywood, NC 35.724 83.087 854 LC218213 LC218264 52 142 Laurel Top 45 Sevier, TN 35.664 83.328 1,680 Everaerts et al. 2008 AB424883 AB425905 53 143 Dry Sluice Gap 45 Sevier, TN 35.638 83.369 1,655 LC218214 LC218265 54 148 Balsam Point 45 Sevier, TN 35.653 83.479 1,469 LC218215 LC218266 55 149 West Point 45 Sevier, TN 35.657 83.450 1,814 LC218216 LC218267 56 150 LeConte North 45 Sevier, TN 35.665 83.439 1,653 LC218217 LC218268 57 151 Trillium Gap 45 Sevier, TN 35.674 83.433 1,439 LC218218 LC218269 58 152 Grotto Falls 45 Sevier, TN 35.674 83.449 1,106 LC218219 LC218270 59 161 Double Spring Gap 45 Swain, NC 35.564 83.534 1,332 LC218220 LC218271 4 Journal of Insect Science, 2017, Vol. 17, No. 3

Fig. 1. Phylogenetic relationships of the Cryptocercus punctulatus species complex distributed in Great Smoky Mountain National Park based on the mitochondrial COII gene sequences (448 bp). The topology and branch lengths shown were obtained by Bayesian inference method. Posterior probabilities (PP) are shown above branches to indicate the level of support for each node. Only one number is given if PP was identical at that node in the two different runs. Numbers below each branch indicate the percentage of bootstrap support (1,000 replicates) in the maximum likelihood (ML) and maximum parsimony (MP) methods, respectively. An asterisk indicates a node that was not supported by ML and MP methods. Clades labels I–VI correspond to those in Figures 2 and 3b. software Mac Multivariate Analysis ver. 2.0 (Esumi, Tokyo, Japan). In both phylogenetic trees (Figs. 1 and 2) the sampled cock- On the basis of the PCA analysis, we compared elevation difference roaches split into two major clades: one that included C. punctulatus and genetic distance (using COII sequence divergence based on the with chromosome counts of 2n ¼ 37 and 2n ¼ 39, and one that T92 þ G model) between two locations within each phylogenetic included those with chromosome counts of 2n ¼ 45. All cockroaches group. Pearson’s correction coefficient was calculated using with a chromosome count of 2n ¼ 45 were clearly monophyletic in Microsoft Excel for Mac 2011 ver. 14.7.2. P values < 0.05 were both the COII and ITS2 trees. In the clades that include the 2n ¼ 37 considered statistically significant. and 2n ¼ 39 karyotypes, the chromosome counts of individuals from two locations were at odds with the molecular signatures used to construct the trees. Collection #106 (Sunkota Ridge) had a chromo- Results some count of 2n ¼ 39 but fell into the 2n ¼ 37 clade in both the Karyotype and Molecular Analysis COII and ITS2 analyses. Just one of the three slides made from testes As previously reported (Nalepa et al. 2002, Everaerts et al. 2008), of adult males from this location was suitable for chromosome anal- the 2n ¼ 37, 39 and 45 karyotypes were found within the boundaries ysis; counts of chromosomes of meiotic cells on that slide were made of GSMNP; more intense sampling did not result in the discovery of independently by the first and last authors and both came to the con- additional karyotypes of C. punctulatus within the park. clusion of 2n ¼ 39. The second discrepancy was collection #113 Journal of Insect Science, 2017, Vol. 17, No. 3 5

Fig. 2. Phylogenetic relationships of the Cryptocercus punctulatus species complex distributed in Great Smoky Mountain National Park based on the nuclear ITS2 sequences (424 bp including gaps). The topology and branch lengths shown were obtained by Bayesian inference method. Note that this tree is unrooted. Posterior probabilities (PP) are shown above branches to indicate the level of support for each node. Only one number is given if PP was identical at that node in the two different runs. Numbers below each branch indicate the percentage of bootstrap support (1,000 replicates) in the maximum likelihood (ML) and maximum parsimony (MP) methods, respectively. An asterisk indicates a node that was not supported by ML and MP methods. Clades labels I–VI correspond to those in Figures 1 and 3b.

(Collins Creek Trailhead), which had a chromosome count of are the two clades of the 2n ¼ 45 karyotype group (clades V and VI: 2n ¼ 37, but grouped with the 2n ¼ 39 clade in both trees. In this Fig. 3b). All three karyotypes potentially come into contact in the location all three chromosome preparations were suitable for chro- middle of the park, in the high-elevation area between Newfound mosome counts, and were consistently 2n ¼ 37. Gap and Clingmans Dome. The present results support the suggestion that the different kar- Distributions yotypes are not separated along altitudinal gradients (Nalepa 2003). The 2n ¼ 45 karyotype is found almost exclusively in the Tennessee The elevation of the 2n ¼ 37 karyotype in GSMNP ranged from 317 half of the park and along the ridgeline separating Tennessee from to 1,895 m; 2n ¼ 39 ranged from 711 to 1,505 m, and 2n ¼ 45 North Carolina (Fig. 3). The four sites with this chromosome num- ranged from 507 to 1,932 m. Each karyotype, then, is represented in ber found in North Carolina are all located on the eastern boundary the range of elevations found in the park, 300 m to over 1,800 m of GSMNP (sites # 39, 90, 137, 138). The karyotype 2n ¼ 39 forms (Nichols and Langdon 2007). a fairly cohesive group in the southeastern quadrant except for the Estimation of Divergence. Using rates of 0.13–0.30%/Myr and intrusion of the karyotype anomaly at site #113. The distribution of mean pairwise TV distances, clades I–IV (male 2n ¼ 37 or 39) and the 2n ¼ 37 group appears to wrap the boundary of the park except V þ VI (male 2n ¼ 45) diverged 8.83–20.38 Myr ago (mean in the easternmost quarter; it also may be common in the interior of TVs ¼ 2.65%, n ¼ 864; Table 2). Among the I–IV groups, diver- the largely un-sampled western third of the park. The three distinct gence events between I and III (male 2n ¼ 37) and IV (male clades of the 2n ¼ 37 karyotype in the COII and ITS2 phylogenetic 2n ¼ 39), and I þ II and III (within male 2n ¼ 37) occurred almost at trees are also geographically distinct (clades I, II and III: Fig. 3b), as the same time (2.29–5.29 Myr ago, mean TVs ¼ 0.69%, n ¼ 231 and 6 Journal of Insect Science, 2017, Vol. 17, No. 3

Fig. 3. Map of karyotype groups of the Cryptocercus punctulatus species complex in Great Smoky Mountains National Park, North Carolina and Tennessee, USA. (a) Location of sample collections; site numbers correspond to those listed in Table 1. (b) Geographic distribution of clades I–VI as indicated in Figures 1 and 2. The white line indicates the ridgeline between North Carolina and Tennessee.

80, respectively). On the other hand, I and II (within male 2n ¼ 37) and the sampling locations (latitude, longitude and elevation shown diverged relatively recently (1.29–2.97 Myr ago, mean TVs ¼ 0.39%, in Table 1). The first principal component accounted for 51.25% of n ¼ 63), and divergence events between V and VI (within male the total variance, and positively reflected chromosome numbers 2n ¼ 45) occurred 0.46–1.06 Myr ago (mean TVs ¼ 0.14%, n ¼ 92). and phylogenetic groups (eigenvector: 0.54 and 0.55, respectively). Within sampling locations, elevation had a higher eigenvector (0.31) PCA than latitude (-0.02) or longitude (-0.07). We then compared eleva- PCA was performed using the chromosome numbers (2n ¼ 37, 39, tion difference and genetic distance (using COII sequence divergence 45), the phylogenetic groups obtained (I–VI shown in Figs. 1 and 2) based on the T92 þ G model) between two locations within each Journal of Insect Science, 2017, Vol. 17, No. 3 7

Table 2. Estimated divergence times between each clade inferred hydrocarbon profiles is not yet clear. Although there are disparities from COII gene sequences (Figs. 1, 3b) between hydrocarbon profile and chromosome number in other parts of the range, in the current study they are concordant in the Comparison Mean 0.3%/Myr 0.13%/Myr TVs (%) sites in which hydrocarbon profiles are known (Everaerts et al. 2008). In four sites of 2n ¼ 45 that have been analyzed for cuticular I vs. II (n ¼ 63) 0.386 1.287 2.969 hydrocarbons (#114, 126, 134, 142), all fall into the same hydrocar- I þ II vs. III (n ¼ 80) 0.687 2.290 5.284 bon group (HcG IV), as do those of the 2n ¼ 37 karyotype (#122, I þ II þ III vs. IV (n ¼ 231) 0.687 2.290 5.285 123, 129; HcG II). The one site of the 2n ¼ 39 karyotype group that V vs. VI (n ¼ 92) 0.138 0.460 1.061 was analyzed (#141) fell into the HcG V group. Analysis of cuticular I þ II þ III þ IV vs. 2.649 8.831 20.380 hydrocarbons of insects from the two sites with karyotype- VþIV (n ¼ 864) molecular disparities (#106 and 113) would be informative. TV ¼ transversion distances. That genetically different clades may have the same karyotype suggests that DNA-sequence changes do not take as long to become phylogenetic clade (I–VI). The results indicate that genetic distance established as do karyotype changes (e.g., clades V and VI both have was significantly correlated with elevation within two clades: clade 2n ¼ 45). This may be because the first-generation progeny of an II (2n ¼ 37) and clade V (2n ¼ 45; Fig. 4). individual with the initial karyotype change would be karyotypic heterozygotes, and may suffer a loss of fertility as a result of irregu- larities in meiosis. Only when two such heterozygotes interbred, Discussion producing new karyotypic homozygotes among their progeny, Incongruence Between Karyotype and Molecular Analyses would normal meiotic pairing and normal fertility be restored in a The karyotype and molecular analyses were largely congruent, with new sub-population. Changes in DNA sequences, on the other hand, two exceptions. Cockroaches from site #106 (Sunkota Ridge) had a may be neutral in their consequences (ITS2 sequences are not trans- 2n ¼ 39 chromosome count but a molecular profile of the 2n ¼ 37 lated into protein), or possibly even have some selective advantage group, and site #113 (Collins Creek Trailhead) was the opposite: in the heterozygous condition (e.g., COII sequences could affect 2n ¼ 37, but it grouped with the 2n ¼ 39 clade in the COII and ITS2 energy metabolism and therefore have relevance for adaptations to trees. Both sites are in the central region of the North Carolina side of temperature during glacial versus interglacial periods). the park, where populations of the two karyotype groups are in close Geographic Distribution and Biogeography of Karyotypes. The contact and may interdigitate or overlap. There is therefore the possi- results indicate that the different karyotypes are currently geograph- bility of sampling error, because in both cases the chromosome counts ically adjacent, with fairly abrupt transitions in their natural contact and the molecular analyses were done on different individuals. zones and no apparent geographic barriers between them. Karyotype groupings are suspected to meet or overlap on very fine spa- Interactions between cockroaches of different karyotypes are there- tial scales; for example, individuals of the 2n ¼ 39 and 2n ¼ 43 karyo- fore inevitable. Lineage boundaries may be maintained by general types (inferred, however, from mtDNA sequencing data) have been reproductive incompatibility between karyotype groups; however, reported from the same log in the southeastern corner of Tennessee different hydrocarbon groups of the same karyotype (see Everaerts (Garrick 2016), although that location is nowhere near the currently et al. 2008), and widely separated populations or clades within the known distribution of the 2n ¼ 43 group. The closest to that Tennessee same karyotype also may be infertile. The analysis of Everaerts et al. site that a 2n ¼ 43 karyotype (based on actual chromosome counts) is (2008), for example, indicates the 2n ¼ 43 karyotype group is not a known to occur is 230 km northeast in Yancey Co., NC, and 280 km monophyletic group, based on mtDNA, nuclear DNA, and cuticular east-northeast in Burke Co., NC (Nalepa et al. 2002). Further work is hydrocarbons. required to determine if the Tennessee finding represents an isolated, An open question is whether these geographic distributions are disjunct population of that karyotype. The 2n ¼ 43 karyotype was not currently stable or are moving as one group invades areas previously found in GSMNP during this study. occupied by another. It is relevant in this regard that in their broader Alternative explanations to possible sampling error are that the range, the distribution of 2n ¼ 39 group is split by a population of karyotype discrepancies in this study—one 2n ¼ 39 individual in a 2n ¼ 37. It is the northern population of the 2n ¼ 39 karyotype 2n ¼ 37 clade, and a 2n ¼ 37 individual in a 2n ¼ 39 clade—might group that is in GSMNP; another population is known from the be explained either as the result of occasional Robertsonian chromo- extreme southwest corner of North Carolina extending into north- somal rearrangements (of the same kind that produced the karyo- ern Georgia (Nalepa et al. 2002: Fig. 2). These two populations are typic population-differences in the first place) or, more likely, as separated by a swath of the 2n ¼ 37 karyotype group that is contigu- first- or second-generation hybrids between different chromosomal ous with sites #49 and #50 reported here. populations. As noted, both of these “tree-discrepant karyotypes” were We can only speculate on the relative importance of the multi- found in locations that could be boundary- or overlap-regions between ple biotic, abiotic and historical factors that created the present the different karyotypes. Ongoing studies on Cryptocercus have shown day geographic pattern, as it is the result of the complex interaction that crosses between different karyotypes sometimes produce viable of intrinsic, lineage-specific traits and the exogenous factors that offspring (Nalepa, Maekawa and Luykx, in prep.). There is evidence affect their host logs on both an ecological and evolutionary scale. for at least partial reproductive compatibility between naturally occur- It has long been thought that the distribution of the genus ring karyotypic variants in other insects, as well as in fish and mam- Cryptocercus is linked to its limited capacity for dispersal mals (Capanna et al. 1976, Luykx and Syren 1981, Herzog and (Mamaev 1973), a feature associated with regional endemism, con- Harrington 1991, Castiglia and Capanna 1999, Choochote et al. 2002, servation of patterns of genetic variation, easily fragmented distri- Tanuja et al. 2003, Pazza et al. 2006, Horn et al. 2012). butions, and the tendency to form parapatric boundaries (Bull Taxonomic subdivision of cockroaches has been indicated by 1991, Cruzan and Templeton 2000, Yeates et al. 2002, Grove cuticular hydrocarbons (Brown et al. 1997, Everaerts et al. 2008), 2002). The genus is wingless, so movement of adults and large but in the case of Cryptocercus the significance of different cuticular nymphs is limited by how far they can walk (Nalepa and Grayson 8 Journal of Insect Science, 2017, Vol. 17, No. 3

Fig. 4. Genetic distance as a function of elevational differences within each clade indicated in Figures 1, 2 and 3b. Genetic distance was calculated from COII sequence divergence based on the T92 þ G model used in the phylogenetic analysis. The square of Pearson’s correction coefﬁcient (R2) and P values (*<0.05 and **<0.01) are shown at the upper right of each graph.

2011). The distance they range is unknown, but most adults within In undisturbed, mature forests of GSMNP the cockroaches likely a population outbreed (Yaguchi et al. 2016). There is no evidence find what they need within a modest ambit, as there is a relative that cockroaches of any one karyotype have a better dispersal stability of tree mortality and deposition of CWD to the forest floor capacity than others. Another relevant feature of the insect is its in old growth temperate forests (Davis et al. 2015). Furthermore, low population growth linked to semelparity and an unusually accumulation of CWD in GSMNP is high for the region, particularly long generation time for an insect. The time from hatch to hatch in in cove forests; this is attributed to large bole size of canopy trees the 2n ¼ 43 population at Mountain Lake Biological Station, and their tendency to suffer higher mortality (Busing 2005 and refs. Virginia, is 5–6 years (Nalepa et al. 1997: Table 2); consequently, therein). Mature forests, however, tend to be patchworks in varying there have been only 13 or 14 generations since GSMNP became a stages of succession due to both natural and anthropogenic distur- national park in 1934. bances (Harmon et al. 1986). Insects, disease, fire, wind throw, and The survival, reproduction and development of Cryptocercus ice storms can devastate entire stands and result in pulses of CWD depends on the presence of moist, rotted logs, with the log host- that serve as habitat for Cryptocercus. In recent history, exotic pest range determined by the tree species composition of the inhabited species have been particularly significant in altering forests and forest (Cleveland et al. 1934, Nalepa and Bandi 1999, Nalepa CWD in the park (Davis et al. 2015, Tuttle and White 2016). The 2003). The cockroach is found in CWD on the floor of all major for- American chestnut [Castanea dentata (Marsh.) Borkh.], for exam- est types in GSMNP, and current evidence suggests that cockroaches ple, covered a full 31% of the Smokies in the early 1900s, grew to with different karyotypes are ecologically interchangeable. This lack mammoth sizes (Brown 2000), and served as important habitat for of host–plant specificity has strong implications for the distribution Cryptocercus (Cleveland et al. 1934, Hebard 1945). When that spe- of the insect in both ecological and evolutionary time. Specifically, cies was decimated by chestnut blight [Cryphonectria parasitica within its range the location of the insect is determined by the pres- (Murrill) Barr.], there was a shift in trees that dominated the canopy ence of forest and the dynamics of CWD deposition, accumulation (Elliot and Swank 2008), and Cryptocercus had no problem in mov- and depreciation; the vegetational composition of the forests is of lit- ing into logs of alternative species on the forest floor (Nalepa 2003). tle relevance. A more recent example is the death of Fraser firs [Abies fraseri Journal of Insect Science, 2017, Vol. 17, No. 3 9

(Pursh) Poir] from an infestation of balsam woolly adelgid [Adelges There would have been a time lag before Cryptocercus could piceae (Ratzeburg)]; this has resulted in estimates of 70–90% mor- repopulate high elevation sites when the climate began to warm, as tality in GSMNP, with a magnitude of CWD among the highest there is a disequilibrium between climate change and forest expan- reported in the literature for the eastern US (Houk 1993, Smith and sion with the latter notably delayed because vegetation zone margins Nicholas 1998, Rose and Nicholas 2008). Similar cases include the migrate only when seeds are blown into the treeless areas (Pielou death of hemlock from hemlock woolly adelgid (Adelges tsugae 2008). Additional time would lapse before the migrating forests Annand; Krapfl et al. 2011), and the near simultaneous death of pine reached a successional stage where CWD suitable for Cryptocercus stands across GSMNP between 1999 and 2003 from southern pine would be present; some ecologists estimate that it takes 200– beetle (Dendroctonus frontalis Zimmerman; Webster and Jenkins 1,000 years for a forest to achieve climax in the Appalachians 2005). Biological disturbances such as these provide bonanzas of host (Constantz 2004). Recolonization of the cockroaches into a tree- material for the cockroach, suggesting that local populations would decimated area is therefore a prolonged downstream effect of cli- take advantage of the surfeit of woody material and remain in a given mate and topography in allowing reforestation and successional area as long as other tree species eventually filled gaps in the canopy, climax in a region previously denuded of vegetation. allowing the abundant CWD to remain suitably moist. Cryptocercus In the opposite scenario, the extinction or exodus of the cock- can cross gaps in standing vegetation as long as some large logs remain roaches from areas where trees have declined because of a severely on the substrate in treeless areas (see Fig. 10.5 in Bell et al. 2007). cooling climate also would be associated with a time lag. In this Anthropogenic disturbances, on the other hand, typically involve case, however, the lag is associated with the pace of accumulation the removal and destruction of CWD. The Great Smoky Mountains and then degeneration of CWD: a boom in resources as a large num- were clear-cut, burned, mined, settled, farmed, and grazed for more ber of trees died, then a bust as the logs cycled through the decom- than a century before the area was established as a national park; position process. Log residence has been estimated at 46–124 years, much of the original forest cover was lost because of human activity depending on a variety of factors (Davis et al. 2015 and refs. (Pyle 1985, Houk 1993, Pierce 2000, Brown 2000, Linzey 2008). therein), one factor undoubtedly being Cryptocercus itself. Large- Although the park still contains a large tract of primary forest, bodied saproxylic insects such as Cryptocercus accelerate log 80% is second growth recovering from logging and settlement, decomposition by channeling and fragmenting dead wood, and via particularly in its western half (Jenkins 2007, Tuttle and White their biotic interactions with microbes (Hanula 1993, Ulyshen 2016). Some sites remain without continuous forest cover >90 years 2016). It has been suggested that Cryptocercus may pulverize logs after logging, and large diameter CWD is least common in forests on a time scale exceeding that of termites (Bell et al. 2007: Fig 10.1). with a history of concentrated settlement (Webster and Jenkins The C. punctulatus species complex dominates the saproxylic 2005, Tuttle and White 2016). In this study, the central regions of guild in GSMNP and occupies the same niche as does the subterra- the park where the three karyotype groups of C. punctulatus meet nean termite Reticulitermes spp. at lower elevations (Nalepa et al. are in or near areas that were not logged intensively and are there- 2002). Like termites (Collins 1989), these cockroaches have the fore high in virgin-forest attributes (Pyle 1985). potential to influence forest productivity by their effect on soil fertil- On an evolutionary scale, the divergence of different karyotype ity. Cryptocercus produces abundant, large, fecal pellets, and their groups of the C. punctulatus species complex probably occurred gut microbiota fix nitrogen at rates comparable to those of termites prior to the Pleistocene glaciations, making it difficult to infer the on a body-weight basis (Breznak et al. 1974, Breznak 1975, Tai sequence of events leading to their present distribution. The most et al. 2016), thus providing a mechanism for nitrogen return to the recent estimate of the genetic structuring of the Appalachian popula- ecosystem (Nardi et al. 2002). In sum, Cryptocercus plays a critical tions of Cryptocercus is at 18 mya, with the 2n ¼ 37 and 39 karyo- role in decomposition of dead wood, contributes to soil formation types diverging 10 mya (Che et al. 2016). There have been an and nutrient cycling (Speight 1989), and should be considered a estimated 18–20 glaciations in the last two million years (Constantz long-term stabilizing force in the forest ecosystems of GSMNP. 2004), and with each successive glacial cycle the species complex has apparently undergone range contraction and isolation, followed by expansion and secondary contact, potentially overriding or eras- Acknowledgments ing previous distributional patterns. The current pattern suggests We thank Patrick Rand and Elaine Glowniak for help with sampling the that during the last glacial maximum, when high elevations in the insects, Hajime Yaguchi, Nako Hatsumura, and Keima Kai for help with park were above treeline (King and Stupka 1950, Whittaker 1956, molecular work, Thomas Colson and Megan Nelson of the National Park Watts 1980, Wright 1987, Delcourt and Delcourt 2000), clades Service for mapping the collection locations, and Becky Nichols of the within two karyotype groups examined in this study (I and II: National Park Service for information and guidance. The work was supported 2n ¼ 37, and V and VI: 2n ¼ 45) may have harbored in separate ref- in part by JSPS KAKENHI Grant Number JP16K07511 to Kiyoto Maekawa. ugia in ravines or slope habitats adjacent to river valleys on opposite The comments of reviewers substantially improved the manuscript. sides of the ridgeline in central GSMNP. 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